Pseudomonas taiwanensis VLB120 toward accumulation of L-phenylalanine and its conversion into the chemical building block t-cinnamate. We recently reported rational engineering of Pseudomonas toward L-tyrosine accumulation by the insertion of genetic modifications that allow both enhanced flux and prevent aromatics degradation. Building on this knowledge, three genes encoding for enzymes involved in the degradation of L-phenylalanine were deleted to allow accumulation of 2.6 mM of L-phenylalanine from 20 mM glucose. The amino acid was subsequently converted into the aromatic model compound t-cinnamate by the expression of a phenylalanine ammonia-lyase (PAL) from Arabidopsis thaliana. The engineered strains produced t-cinnamate with yields of 23 and 39% Cmol Cmol−1 from glucose and glycerol, respectively. Yields were improved up to 48% Cmol Cmol−1 from glycerol when two enzymes involved in the shikimate pathway were additionally overexpressed, however with negative impact on strain performance and reproducibility. Production titers were increased in fed-batch fermentations, in which 33.5 mM t-cinnamate were produced solely from glycerol, in a mineral medium without additional complex supplements. The aspect of product toxicity was targeted by the utilization of a streamlined, genome-reduced strain, which improves upon the already high tolerance of P. taiwanensis VLB120 toward t-cinnamate.Microbial biocatalysis represents a promising alternative for the production of a variety of aromatic chemicals, where microorganisms are engineered to convert a renewable feedstock under mild production conditions into a valuable chemical building block. This study describes the rational engineering of the solvent-tolerant bacterium  Trans-cinnamate is an aromatic compound naturally occurring in plants, where it serves as central intermediate for the biosynthesis of a large number of substances, including coumarins, flavonoids, and phenylpropanoids   agar]. After tri- or four-parental mating procedures, Pseudomonads were isolated on cetrimide agar (Sigma Aldrich) plates supplemented with 10 mL L−1 glycerol. Kanamycin (50 μg mL−1) or gentamicin (20 μg mL−1) was added to cultures or plates when necessary.Strains and plasmids used in this study can be found in P. taiwanensis were grown in mineral salt medium (MSM) adapted from Hartmans et al. (2HPO4 and 68 mM NaH2PO4). Glucose or glycerol were added as sole carbon source at indicated concentrations. Main cultures were inoculated at an OD600 of 0.2, from seed cultures grown in MSM containing glucose as carbon source. Production experiments were performed in 500 mL Erlenmeyer flasks with a culture volume of 50 mL, which were cultivated in a rotary shaker with a frequency of 200 rpm and a throw of 50 mm. Toxicity assays were performed in 96-round low-well plates with a filling volume of 200 μL in the System Duetz® cultivation system  with a shaking frequency of 225 rpm. OD600 values were calculated from the measured green values using a calibration. Phenylalanine accumulation was analyzed in 24-square deep-well plates with a filling volume of 5 mL and a shaking frequency of 225 rpm, and growth was monitored in the Growth profiler® system.During production and toxicity experiments in shake flasks and well plates, liquid cultures of s et al.  at pH 7.aroGfbr (from E. coli K12 W3110) and AtPAL  were codon-optimized for P. taiwanensis VLB120 using the online tool OPTIMIZER . Integration at the attTn7-site was achieved by patch-mating of the E. coli donor strain holding the respective pBG-plasmid, the helper strain E. coli HB101 pRK2013, DH5α λpir pTNS1 providing the required transposase and the recipient Pseudomonas taiwanensis strain as described by Wynands et al. .For plasmid construction, DNA fragments were PCR amplified using the Q5 high-fidelity polymerase  with corresponding overhangs to enable subsequent Gibson assembly . The starting volume was 100 mL and the temperature was maintained at 30°C. Gas supply was provided via headspace with a starting gas flow rate of 6 sL h−1. The initial agitation frequency was 500 rpm. The dO2 was controlled with a cascade, keeping the concentration of diluted oxygen at 35% by first increasing the agitation speed up to 1,200 rpm, then increasing the oxygen concentration in the air supply to a maximum of 80% and subsequently increasing the gas flow rate. pH 7 was maintained by automatic addition of 5% NH3 or 1 M H2SO4. Cells were grown in MSM according to Hartmans et al. . The initial batch medium contained either 20 mM of glucose or 40 mM of glycerol as sole carbon source. The reactors were operated in a dO2-controlled fed-batch mode. When glucose or glycerol was depleted, the dO2 signal rapidly increased as a result of metabolic arrest. A dO2 signal >70 triggered a feed pump, resulting in a feed shot of 5 mM of glucose or 10 mM of glycerol per trigger initiation.Fermentations were carried out in fed-batch operation mode in DASbox® mini-bioreactors using the DASware control software . The reactors were set up of 385 ml glass vessels, two Rushton-type impellers driven by direct overhead drives, feeding lines for acid, base, and carbon source, a temperature sensor, an EasyFerm Plus K8 120 pH-sensor  and an InPro® 6800 series Os et al. , where tOptical densities of liquid cultures were measured at 600 nm using an Ultrospec 10 Cell Density Meter .−1. Elution took place with a gradient starting at 90% H2O containing 0.1% (v/v) TFA and 10% methanol. This ratio was held for 2 min, followed by gradual increase to 100% methanol over the course of 8 min. After 2 min at 100% methanol, initial ratios were reached again within 1 min and held constant for further 2 min. UV detection was conducted at a wavelength of 245 nm. L-phenylalanine and L-tyrosine quantification shown in 2SO4 at an isocratic flow of 0.5 mL min−1 and a temperature of 30°C for 20 min. Glucose and glycerol were analyzed using the RI detector, gluconate concentrations were determined with the UV detector at a wavelength of 210 nm. Due to co-elution of glucose and gluconate, glucose concentrations were determined by subtraction of the gluconate concentration.Samples taken from the cultures were centrifuged at 13,000 rpm for 2–5 min and the supernatant was analyzed by HPLC. Aromatics quantification was performed using a Beckman System Gold 126 Solvent Module with a 168 diode 201 array detector  and an ISAspher 100-5 C18 BDS reversed-phase 202 column  at 30°C and a flow rate of 0.8 mL mint-cinnamate is mildly toxic at neutral pH and it exhibits a different mode of action. Increasing concentrations of t-cinnamate lead to enhanced osmotic stress in cell cultures, but will also cause the inhibition of specific enzymes and disturb cellular processes, resulting in growth defects  and completely inhibited at >5.4 mM (800 mg L−1) when t-cinnamate is added at the beginning of cultivation  t-cinnamate in MSM with a rate of 0.24 ± 0.01 h−1. P. taiwanensis VLB120 is equipped with particular mechanisms that enable this superior tolerance toward a variety of toxic compounds. While RND-type efflux pumps mainly act on hydrophobic aromatic solvents and a number of antibiotics , or the regulatory genes ttgVW are included to induce the pump in response to solvents (GRC3). The similar performance of GRC1 and GRC3 in the presence of t-cinnamate indicates that this aromatic acid does not induce expression of the ttgGHI, nor does the TtgGHI pump contribute to cinnamate tolerance. Given this similar performance, and in light of the potential to use a t-cinnamate-producing strain as platform for a variety of further industrially relevant hydrophobic aromatics, the strain GRC3 was chosen as host to maintain the possibility of solvent efflux by TtgGHI. As shown in P. taiwanensis GRC3 can grow in the presence of t-cinnamate concentrations of up to 50 mM (7.4 g L−1), which is 10-fold higher than MIC for E. coli. The native tolerance potential of P. taiwanensis VLB120 in combination with improved bioprocess features obtained by streamlining of this strain hence set an ideal starting point for an efficient microbial production process of t-cinnamate.To further exploit the native potential of innamate . The strt-cinnamate is the deamination product of L-phenylalanine and the enhanced supply of this precursor in microbial chassis strains is thus crucial to allow efficient production. A variety of approaches toward L-phenylalanine overproduction were reported over the last years, delivering highly productive strains of e.g., E. coli (63 g L−1 from glucose)   associated to the degradation of shikimate pathway-derived compounds resulted in strains unable to catabolize L-tyrosine, L-phenylalanine, p-hydroxybenzoate, and 3-dehydroshikimate to ensure aromatics accumulation. The flux toward L-tyrosine was subsequently enhanced by the insertion of point mutations into the native locus of genes in the shikimate pathway, resulting in enzymes with amino acid substitutions . The deletion of pykA (encoding for a pyruvate kinase) additionally reduced the flux of PEP toward pyruvate, thereby increasing the precursor pool for the shikimate pathway. An introduction of these mutations in the genome-reduced strains of P. taiwanensis VLB120 led to the efficient production of phenol from L-tyrosine  phhAB encoding for phenylalanine-4-monooxygenase involved in the conversion of phenylalanine to tyrosine  or not connected to the central carbon metabolism (PVLB_10925) under the applied conditions, even though this strain contains the genetic inventory for the full degradation of the resulting 2-phenylacetamide and phenylethylamine. This is underlined by similar observations in an L-phenylalanine overproducing strain of P. putida DOT-T1E  accumulated 3.0 ± 0.07 mM of L-phenylalanine after 70 h. Also, L-tyrosine production was reduced, likely due to PheAT310I increasing the flux of prephenate toward L-phenylalanine.L-Tyrosine and L-phenylalanine production could be increased further by the additional overexpression of AroGP. taiwanensis GRC3 Δ8ΔpykA-tap was subsequently used as host for the production of t-cinnamate. The deamination of L-phenylalanine into t-cinnamate was achieved using the PAL2 enzyme from A. thaliana. While four genes encoding PAL have been identified in A. thaliana , as a result of the reduced growth rate. While this strain has a very high yield, its reliable application as a production host was problematic. When the experiment described above was repeated, the cultures displayed varying growth behavior in terms of lag-phase, growth rates, and productivity  and glycerol (0.11 ± 0.00 mM h−1) compared to the strain additionally expressing aroGfbr and pheAT310I. In the course of the cultivations, no accumulation of L-phenylalanine was observed. This indicates that PAL activity is not inhibited by the concentrations of t-cinnamate reached in these cultures, in contrast to observations in other hosts and with PALs from different organisms  and a volumetric productivity of 0.19 ± 0.20 mM h−1. As shown in 600 of 12.7 after around 65 h. At this point, a decrease in biomass concentration was observed. Furthermore, there was no significant increase in t-cinnamate titers from this point on, in spite of the fact that the base strain GRC3 tolerated higher concentrations of t-cinnamate up to 50 mM  have been reported in fed-batch fermentations of t-cinnamate-producing strains of E. coli (Bang et al., P. taiwanensis GRC strains enables growth without the addition of complex supplements. However, tolerance mechanisms for t-cinnamate remain to be further investigated and exploited to enhance titers and thus the feasibility of microbial t-cinnamate synthesis.In contrast to the strain overexpressing AroGntations . To the P. taiwanensis VLB120 towards efficient t-cinnamate production. The plasmid-free strain bearing no auxotrophies synthesized t-cinnamate from glucose or glycerol, with yields of up to 48% Cmol Cmol−1 in shake flask cultivations. Titers were increased up to 33.35 mM in fed-batch fermentations using glycerol as sole carbon source. As product titers achieved in this study impair fitness and productivity of the chassis strains, the native tolerance features of Pseudomonas allowing enhanced t-cinnamate tolerance require further investigation and enhancement to increase process efficiency. One promising target is the ABC transporter Ttg2ABC, an extrusion pump involved in p-coumarate tolerance (Calero et al., t-cinnamate, its regulation however remains to be investigated. Fine-tuning of additional aroG and pheA overexpression, e.g., by using weaker or inducible promoters, could furthermore avoid the observed growth defects while still maintaining the high yields achieved with this setup. Overall, the results underline the high potential of Pseudomonas species to produce chemical building blocks using aromatic amino acids as precursors. The establishment of efficient microbial production of the model compound t-cinnamate will in future serve as foundation to expand the product collection of this versatile species, ranging from bulk chemicals such as styrene (Lee et al., In this study, we describe the rational engineering of All datasets generated for this study are included in the article/NW conceived and supervised the study with the help of LB. MO and MF performed the experiments with support of BW and CL. BW provided the GRC strains with deletions for L-tyrosine accumulation and deletion vectors. MO wrote the manuscript with the help of BW and NW. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this study, we found that bacteria belonging to Micrococcales and Corynebacteriales exhibit light-dependent carotenoid-like pigment production including an amino acid-producer Corynebacterium glutamicum AJ1511. CrtR is a putative MarR family transcriptional regulator located in the divergent region of a carotenoid biosynthesis gene cluster in the genome of those bacteria. A null mutant for crtR of C. glutamicum AJ1511 exhibited constitutive production of carotenoids independent of light. A complemented strain of the crtR mutant produced carotenoids in a light-dependent manner. Transcriptional analysis revealed that the expression of carotenoid biosynthesis genes is regulated in a light-dependent manner in the wild type, while the transcription was upregulated in the crtR mutant irrespective of light. In vitro experiments demonstrated that a recombinant CrtR protein binds to the specific sequences within the intergenic region of crtR and crtE, which corresponds to −58 to −7 for crtE, and +26 to −28 for crtR with respect to the transcriptional start site, and serves as a repressor for crtE transcription directed by RNA polymerase containing SigA. Taken together, the results indicate that CrtR light-dependently controls the expression of the carotenoid gene cluster in C. glutamicum and probably closely related Actinobacteria.Carotenoid production in some non-phototropic bacteria occurs in a light-dependent manner to protect cells from photo-oxidants. Knowledge regarding the transcriptional regulator involved in the light-dependent production of carotenoids of non-phototrophic bacteria has been mainly confined to coenzyme B They are tetraterpenoids that consist of a polyene hydrocarbon chain derived from eight isoprene units. Most carotenoids consist of 40 carbon atoms that are modified in several ways, such as cyclization and desaturation, to produce a variety of compounds with divergent chemical structures3. Recently, the C30 and C50 biosynthetic pathways were found in Micrococcus, Corynebacterium, and Flavobacterium4. Carotenoids can function as photoprotectors, light harvesting molecules, or membrane stabilizers5. In non-phototrophic bacteria, the main function of carotenoids is the protection of cells from photo-oxidative damage by scavenging harmful agents such as singlet and triplet molecular species produced upon illumination6.Carotenoids are yellow to red colored pigments that are widely produced by plants, algae, and some fungi and bacteria7. We and other groups have been studying the phenomena and molecular mechanism of light-inducible production of carotenoids of phylogenetically different bacteria including S. coelicolor A3(2)8, Thermus thermophilus HB2714, Bacillus megaterium QM B155116, Mycobacterium marinum17, and Arthrobacter arilaitensis18. Our study has revealed that LitR/CarH family proteins, a MerR family transcriptional regulator retained by S. coelicolor, T. thermophilus, and B. megaterium, play a central role in light-dependent gene expression19. Other groups have also reported that LitR/CarH family proteins serve as a photosensor in the light-inducible production of carotenoids in Myxococcus xanthus21.In non-phototrophic bacteria, the control of carotenoid production is classified into three types: constitutive, light-inducible, and cryptic manner-terminus and an adenosyl-coenzyme B12 (AdoB12)-binding domain in its C-terminus, and serves as a negative regulator for light-inducible transcription19. The molecular mechanism of light-inducible transcription mediated by the LitR/CarH family involves the photolysis of AdoB12 associated with LitR/CarH due to light, which results in a large conformational change and inactivation of the DNA-binding activity of LitR/CarH11. LitR homologs are widely distributed in the phylogenetically diverged genera of non-phototrophic bacteria, including gram-negative and gram-positive bacteria, which indicate that LitR/CarH family proteins are general photosensors and have diverse roles in non-phototrophic bacteria. Recently, we have also reported the existence of a novel type of LitR/CarH family proteins in Burkholderia multivorans belonging to Beta-proteobacteria, which requires a different type chromophore from coenzyme B1222.LitR/CarH family proteins commonly contain a DNA-binding domain in its NC. glutamicum, an amino acid producer, exhibit photo-dependent production of carotenoids. The evidence indicates that a light-induced MarR family regulator CrtR, is distributed to some groups of Actinobacteria and is involved in the light-inducible expression of crt biosynthesis genes.The wide distribution of LitR/CarH family protein among non-phototrophic bacteria leads to our assumption that other type of light-inducible transcriptional regulators is present in non-phototrophic bacteria. The finding of a novel type of photosensors would deepen insights on bacterial environmental responses. In this study, we performed a wide screen of bacteria exhibiting light-inducible production of carotenoids, which led us to the finding that some groups of gram-positive bacteria including Bacillus spp. isolated from soil showed light-dependent production of carotenoid on rich LB medium15. Our genetic and biochemical study of B. megaterium QM B1551 revealed that LitR in complex with B12 serves as a photosensitive transcriptional regulator to control the expression of crt biosynthesis genes in a light-dependent manner. Here, we used several media including not only LB but also 10-fold diluted LB medium (1/10 LB), 1/10 LB containing 1% glucose (1/10 LBG), and R2A medium to more widely screen bacteria exhibiting light-inducible yellow-color pigment production . We isolated a number of bacteria from various environmental source including biotope, paddy field, forest soil, bran pickled, snow, residential land, and cattle manure in Japan. Of these, bacteria showing white light-dependent pigment production were subjected to the phylogenetic analysis based on DNA sequence of the 16S rRNA gene. As summarized in Table Micrococcales, a high G + C gram-positive Actinobacteria, including the genus Arthrobacter, Leifsonia, Microbacterium, Brevibacterium, and Agromyces. As minor groups, Bacillus, Sphingobacterium, and Simplicispira genus were also isolated. All isolates exhibited >99% sequence identity with the known species affiliating to those bacterial taxa. This result indicated that 1/10 LB and 1/10 LBG medium were suitable for the isolation of photo-responsive Micrococcales bacteria.We previously reported that a number of Micrococcales obtained from culture collections such as NBRC, JCM, and ATCC. As shown in Fig. Paenarthrobacter, Sinomonas, Glutamicibacter, Isoptericola, Jonesia, and Sanguibacter produced yellow pigments in response to light. To confirm that the pigments are carotenoid, we analyzed the UV-visible absorption spectrum of a methanol extract of the illuminated cells. As shown in Fig. Paenarthrobacter aurescens TC1 (ATCC BAA-1386), Sinomonas atrocyanea NBRC 12956, Isoptericola jiangsuensis JCM 17812, Isoptericola dokdonensis JCM 15137, Jonesia denitrificans DSM 20603, Sanguibacter keddieii DSM 10542. The light-responsive carotenoid production in Arthrobacter arilaitensis RE117 and Mycobacterium marinum M was also reported18. The pigment production in C. glutamicum AJ1511, C. glutamicum ATCC 13032, and C. callunae JCM 9489 was also induced by light , crtEb (NCgl0594), crtYe (NCgl0595), and crtYf (NCgl0596) are known to encode phytoene desaturase, a lycopene elongase, and a carotenoid C45/C50 ε-cyclase23. This result indicated that the yellow-color pigment is a carotenoid, and the ability to sense light was commonly spread in this group of bacteria.To confirm the light-inducible pigment production observed in the isolates, we characterized the phenotype of bacteria belonging to ht Table . The UV- nm Fig. . This prActinobacteria, especially Micrococcales26, Corynebacteriales27, and Streptomycetales7; however, the molecular mechanism underlying the regulation of carotenoid production has not been well characterized except in S. coelicolor A3(2) analyzed in our previous study8. Therefore, we assessed for the gene organization of crt biosynthesis clusters of the genome-sequenced bacteria affiliating with the taxonomic group.It is well known that the carotenoid-producing ability is widespread in groups of bacteria including crt biosynthesis gene cluster  domain protein29 was not found. Alternatively, a MarR family regulator was encoded in the divergent region of the crt gene cluster , A. arilaitensis RE117, Clavibacter michiganensis subsp. michiganensis NCPPB 382, S. atrocyanea NBRC 12956, I. jiangsuensis JCM 17812, I. dokdonensis JCM 15137, J. denitrificans DSM 20603, S. keddieii DSM 10542, Cellulomonas gilvus ATCC 13127, and Brachybacterium faecium DSM 4810 . Therefore, 11 out of 16 strains harboring crtR-crt gene shown in Fig. crt gene cluster.The gene organization consisting of a Micrococcales and Corynebacteriales. As shown in Fig. Micrococcales including the genus Arthrobacter, Leifsonia, Microbacterium, Brevibacterium, and Agromyces, and the order Corynebacteriales including the genus Corynebacterium. The other small branched group includes Mycobacterium and Nocardia belonging to the order Corynebacteriales. CrtRs derived from Corynebacterium were included in the large clade of Micrococcales, which indicates that CrtRs of Corynebacterium and Mycobacterium independently evolved.To classify the CrtR family proteins, phylogenetic analysis was carried out using the full-length amino acid sequences of CrtRs distributed to C. glutamicum AJ1511 (ATCC 13869), an amino acid producer, to examine the light-inducible mechanism. This strain carries advantages of genetic analysis due to its high transformation efficiency as well as the association of carotenoids with unique colony morphologies as shown by our previous study32. In order to examine the physiological role of crtR in C. glutamicum AJ1511 retaining a 97% similarity with that of ATCC 1303227, we generated a crtR null mutant of this strain. In this mutant, a drug marker for selection was removed to exclude possible polar effects on the expression of the flanking coding sequences. The resultant crtR mutant (designated ΔcrtR) exhibited constitutive production of carotenoids under both dark and light conditions . The genetically complemented strain (ΔcrtR/pKMT + crtR) showed similar light-responsive carotenoid production as the wild type . As shown in Fig. crtE and crtR were assigned 85 and 41 nucleotides, respectively, upstream of the translational start codon (TTG for crtE and ATG for crtR). This result was consistent with the start site of crtE in C. glutamicum ATCC 13032 reported previously23. Comparison of each promoter to the consensus sequence recognized with sigma factor revealed that the crtE promoter exhibits a similarity with that of SigA of C. glutamicum, a house-keeping sigma factor. Namely, the potential -35 (5′-TTAAAA-3′) and -10 (5′-TATAAA-3′) sequences of the crtE promoter were similar to those (5′-TTGC/GCA-3′ and 5′-TANAAT-3′) recognized probably by SigA33  cultured under dark and light conditions for 10 and 15 h. The house-keeping 16S rRNA gene was used as an internal control for RNA quality and quantity. As shown in Fig. crtR, BBD29_03565, crtE, and crtI in the wild type was markedly induced under light conditions, while the transcription of phrB encoding a DNA photolyase, and sigA was not largely affected by light. In the ΔcrtR, the transcriptional level of the light-inducible genes was almost identical between the dark and light conditions. The introduction of an intact crtR gene into the chromosome of the ΔcrtR restored the light-dependent transcription of crtR, BBD29_03565, crtE, and crtI. These results were consistent with that for the production of carotenoids, indicating that light-inducible carotenoid production is regulated at a transcriptional level, and CrtR is involved in the control of the crt gene expression. Further, to study the role of CrtR, we calculated the ratios of the transcripts in the ΔcrtR to that in the wild type. As shown in Fig. BBD29_03565, crtE, and crtI were increased in ΔcrtR compared to the wild type. In contrast, the transcripts of phrB and sigA were similar amount in the wild type and ΔcrtR. These results indicate that CrtR serves as a transcriptional repressor to the crt cluster.We performed a quantitative RT-PCR analysis to investigate whether the photo-dependent carotenoid production was regulated at the transcriptional level. For this analysis, total RNA was purified from the wild type, ΔcrtR and crtE to control the light-inducible expression. Therefore, we then performed a gel shift assay to investigate the DNA-binding activity of CrtR to the intergenic region between crtE and crtR. A recombinant protein for CrtR was overexpressed in E. coli and purified to homogeneity by affinity chromatography . As shown in Fig. crtE and crtR in a dose-dependent manner, while the retardation by the CrtR activity was not observed when the sigA promoter region was used as a control probe. The result indicated that CrtR protein specifically binds to the divergent promoter region of crtE and crtR.The above results suggest that the CrtR protein binds to the promoter region of crtE and crtR. As shown in Fig. crtE. These regions corresponded to crtR, +26 to −28 in the sense strand and +25 to −26 in the antisense strand with respect to the TSS, respectively. The determined binding sequence of CrtR overlapped with the −10 and −35 regions of crtE as well as the −10 region of crtR . To examine the specificity of SigA, two DNA fragments with different length were used as templates , which was longer than that of the typical MarR family protein. This suggests that CrtR may bind the multiple sites located in the intergenic region of crtE and crtR, although we could not find inverted repeats. Similar cases with this kind of transcriptional regulator have been known. For example, the length of the binding site of Sulfolobus solfataricus BldR is 40 bp36. PecS of Erwinia chrysanthemi is supposed to different operator sequences as a single or multiple dimers37 within its regulon, thereby demonstrating varying spans of protection from 20 to 100 bp.Recently, Henke ing Figs  and 9. G30. Therefore, we hypothesize that CrtR binds to the promoter region in the absence of an unidentified ligand, and that the binding of its ligand inhibits the DNA-binding activity leading to the expression of crtE. There is a simple hypothesis that blue-light absorption by an unidentified ligand causes its binding to CrtR, which results in the aforementioned derepression and the activation of the crtE promoter. Based on this hypothesis, we examined whether known light-absorbing molecules such as FAD, FMN, para-coumaric acid, retinol, AdoB12 had an effect on the DNA-binding activity of CrtR, but the addition of the light-absorbing molecules did not affect effect on the DNA-binding activity of CrtR (our unpublished data). Recently, it has been reported that GGPP, a precursor of carotenoids, interfered with the in vitro DNA-binding activity of CrtR of C. glutamicum ATCC 1303227, but we have not yet succeeded in reproducing the effect of GGPP on CrtR activity.Generally, apo-MarR-type regulator, a free ligand protein, represses the expression of the target gene by its binding to the operator, while in the presence of ligand, its activity as a repressor is diminished due to the interaction with the ligandBBD29_03565 product belongs to a family of multidrug resistance pumps termed RND  proteins that recognize and mediate the transport of a great diversity of compounds40. In P. aeruginosa, the expression of the multidrug transporter MexAB-OprM, an RND family member, is regulated by MexR, a MarR-type regulator41. Currently, we do not think that BBD29_03565 is involved in carotenoid biosynthesis, since the inactivation of BBD29_03565 did not affect the production of carotenoids and its transcriptional level (our unpublished data). Possibly, it may play a role in the elimination of an unidentified ligand for CrtR, which becomes toxic if illuminated. However, the inactivation of BBD29_03565 did not affect its growth both under light and dark conditions, which suggests that the accumulation of the ligand is not toxic for C. glutamicum. The conservation of CrtR and RND family members in close proximity to each other was also found in the genome of Mycobacterium marinum M .The C. glutamicum and related bacteria. We anticipate that further studies focusing on the ligand for CrtR will elucidate the signaling pathway based on the unique light-responsive regulatory system in C. glutamicum. As evidenced by the previous report that an engineered C. glutamicum exhibited the high productivity of lycopene23, C. glutamicum harbors high potency of this bacterium as a production host of terpenoids. Our findings regarding the light-inducible gene expression may eventually reveal the ability of C. glutamicum and related bacteria to produce novel types of carotenoids and expand the possibility of their industrial application.Although the exact mechanism therein is not yet known, the identification and characterization of the ligand of CrtR will help in understanding the exact mechanism of light-dependent transcriptional control in C. glutamicum AJ1511 (ATCC 13869) was obtained from Ajinomoto . Other bacterial strains  and BL21(DE3)pLysS  were used as hosts for DNA manipulation and protein expression, respectively. The pUC118 and pMD19 (Takara Bio) plasmids were used as general cloning vectors in E. coli. The pK18mobsacB plasmid42 obtained from National BioResource Project , a kanamycin-resistance E. coli vector, was used for gene disruption in C. glutamicum. The pET26b( + ) plasmid (Merck) was used for the overexpression of CrtR and SigA in BL21(DE3)pLysS. The chemicals and enzymes used for DNA manipulation were purchased from Wako , Kokusan , and Takara Bio unless otherwise indicated. For the screening media, Luria-Bertani (LB) containing 1.0% Bacto Tryptone , 1.0% yeast extract , 0.5% NaCl (pH 7.2 by using NaOH), 10-fold diluted LB (pH 7.2) (designated 1/10 LB), 1/10 LB with 1.0% glucose (pH 7.2) (designated 1/10 LBG), and R2A  were used. To prepare the solid medium, 1.5% agar was added. The conditions for the culture and genetic manipulation of E. coli were performed as described previously43. C. glutamicum was grown on CM2B medium containing 1.0% polypeptone , 1.0% yeast extract, and 0.5% NaCl (pH 7.0 by using KOH) or LB medium. To prepare the CM2B solid medium, 2.0% agar was added. Liquid culture was performed in a 500-mL baffled flask containing 100 mL medium in a rotary shaker at 135 rpm. Light irradiation was performed by an illuminating incubator  equipped with a white or blue light fluorescent lamp . Light intensity was measured using a Model Li-250A Light Meter . To select for transformants of E. coli and C. glutamicum, ampicillin and kanamycin were added at 50 μg/ml.ns Table  were obtTo isolate bacteria, 1.0 g soil was suspended in 10 mL sterile distilled water, and incubated at room temperature for 1 h. The supernatant was diluted by distilled water, and spread on solid media including LB (pH 7.2), 1/10 LB (pH 7.2), 1/10 LBG (pH 7.2), and R2A. The single colonies were inoculated to two plates with tooth picks, and cultured under dark and light conditions at 28 °C for 2 or 3 d. The isolates exhibiting light-dependent pigment production were purified by single colony isolation method. A total of 24 strains out of 1,100 isolates exhibited photo-dependent yellow pigment production.http://www.ncbi.nlm.nih.gov/BLAST/) and the SEQUENCE_MATCH program from the Ribosomal Database Project database44.To taxonomically characterize the isolates, total DNA was extracted from the isolates by the PurElute Bacterial Genomic Kit . The PCR amplicons of the 16S rRNA gene using the Eubacterium universal primer pair B8F/B1492R . The amino acid sequences were analyzed using ClustalW45 and MEGA 546. Molecular phylogenetic trees were reconstructed by the neighbor-joining method47. The distance matrix was calculated using Kimura’s two-parameter model48.A neighbor-joining phylogenetic tree based on the full length amino acid sequence of CrtR homologs was constructed. The CrtR amino acid sequences, which are located in C. glutamicum AJ1511 (ATCC 13869) and C. glutamicum ATCC 13032 (JCM 1318) were cultured at 28 °C for 2 days on solid LB medium under dark and light conditions using an illuminating incubator equipped with white-light fluorescent lamps. Under light conditions, the solid culture was illuminated with white light at approximately 2.4 µmol s–1 m–2. The same lamp, covered with a blue-light filter, was used to generate blue (400–460 nm) light. The method of extracting carotenoids was described previously15. The absorption spectrum of the carotenoid fraction was recorded by using a UV spectrometer .crtR was carried out using pK18mobsacB, which has a sacB gene to use as a counter-selective suicide marker42. To delete the crtR gene from the AJ1511 strain, regions approximately 1 kb upstream and downstream of crtR were amplified by PCR using the genomic DNA as a template with the two primer sets DL-F/DL-MR and DL-MF/DL-R . The pDIS plasmid was introduced into C. glutamicum by electroporation32, and the single crossover strains were able to grow on a LB agar plate containing kanamycin. Subsequently, the kanamycin-sensitive derivatives of the single crossover strains that were able to grow on LB agar plates containing 10% sucrose were selected. The expected double crossover-mediated homologous recombination in such derivatives was confirmed by PCR. The resulting kanamycin-sensitive recombinants were assessed to identify true recombinants by performing appropriate PCR and hybridization experiments, and one of the true recombinants was described to be a crtR-null mutant.Construction of a markerless mutant of -R Table . The twoC. glutamicum, we first constructed a chromosome integration vector, pKMT1, for homologous recombination, which retains a partial gene sequence of thrC (NCgl2046), a threonine dehydratase. The internal region corresponding to 113 to 1261 nt of thrC was amplified by PCR using the primer pair thrCF and thrCR  according to the manufacturer’s instruction. The concentration of total RNA was measured with the NanoDrop Lite . The following cDNA synthesis and its quantification with PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) and an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific) was performed according to manufacturer’s manual and our previous study22. For sample normalization, 16S rRNA gene was used as an internal control. The cDNA of sigA, crtE, crtI, BBD29_03565, crtR, and phrB were detected by the primer pairs, sigA-F/sigA-R, crtE-F/crtE-R, 3565-F/3565-R, limR-F/limR-R, and phrB-F/phrB-R, respectively  and by Directed Amplification of TSSs (DMTSS)50, respectively. The 5′-Full RACE Core Set was used according to the manufacturer’s instructions, and DMTSS was performed as described50. In both experiments, 2 µg of total RNA and gene-specific primers (RT-RACE) were used . The resulting plasmid directed the expression of CrtR fused to a C-terminal 6× His-tag in E. coli BL21(DE3)pLysS. The expression and purification of the recombinant followed the standard protocol for the His-tagged protein recommended by the manufacturer. To prepare the recombinant protein SigA (NCgl1836) with a His-tag at its C-terminus, a housekeeping major sigma factor, a protein expression vector for sigA from C. glutamicum ATCC 13032 similar to CrtR, was constructed. The primer set used was SigAex-F/SigAex-R (Table E. coli BL21(DE3)pLysS and purified to near homogeneity by affinity chromatography according to manufacturer’s instruction. The absorption spectra of the resultant recombinants were recorded by using a Cary 60 UV-Vis spectrophotometer (Agilent Technologies) or Multiskan GO spectrophotometer (Thermo Fisher Scientific). The protein concentration was measured with a protein assay kit , and the absorbance was measured with the NanoDrop Lite (Thermo Fisher Scientific).For the expression of CrtR in -R Table . The res-R Table . The SigcrtR-PcrtE, and PA-F/PA-R for PsigA , 0.5 mM EDTA-NaOH (pH 8.0), 50 mM KCl, and 10% glycerol. After incubation at 30 °C for 30 min, DNase I was added at a final concentration of 20 µg/ml, and the mixture was further incubated for 1 min at 25 °C. The reaction was terminated by the addition of 100 µl of the stop solution  and 300 µl of phenol-chloroform (1:1). After ethanol precipitation, the pellet was washed with 80% ethanol, and dissolved in a 6-µl formamide dye mixture. The samples ware applied to a 6% urea-polyacrylamide gel. Maxam-Gilbert sequencing ladders (G + A and T + C reactions) generated from the 32P-labeled probe DNA fragment were used as a reference. The gels were imaged with a Typhoon 9410 image analyzer.To determine the binding site of CrtR, a DNase I footprint analysis was carried out as described previouslyin vitro run-off transcriptional assay was performed as described previously15. To prepare two DNA templates with different lengths, DNA fragments including the intergenic region for crtE-crtR were prepared by PCR with the primer pairs Runoff-F/Runoff-RA  and Runoff-F/Runoff-RB . A commercially available RNA polymerase core enzyme of E. coli  was used because this enzyme has been frequently used in experiments in Streptomyces. The [γ-32P]ATP-labeled Marker 10 (pBR322 cut with MspI)  was used as a size marker. The reaction products were separated by denaturing polyacrylamide gel containing 6% acrylamide and 8 M urea. The radioactive signals detected were similar to those for the DNase I footprint.To analyze the function of SigA and CrtR, an Supplementary Table S1, Figure S1, and Figure S2


To compare swept source OCTA device, with and without the extended field imaging (EFI) technique, to standard fluorescein angiography (FA) in the clinical practice.Consecutive patients with vascular disorder patients underwent FA with 55-degree lens  and OCTA with the prototype PlexElite  using a 12 mm x 12 mm volume scan pattern centered on the fovea and a prototype of + 20.00-diopter designed specifically by Zeiss. The imaging methods were compared for visible field of view, extension of non-perfused areas, presence and number of neovessels, vessel density (VD) and fractal dimension (FD).2) was significantly higher than SS-OCTA without EFI (20.46 ± 18.70 mm2), and with FA (27.55 ± 4.4 mm2). The mean VD and FD of EFI SS-OCTA were significantly different compared to SS-OCT without EFI.Forty-three eyes of 27 patients were included. The mean extension ratio of EFI SS-OCTA compared to SS-OCTA without EFI and FA were 1.97 ± 0.02 and 0.85 ± 0.01. The mean extension of non-perfused areas with EFI SS-OCTA (34.22 ± 33.4 mmEFI SS-OCTA captured larger areas than SS-OCTA without EFI and FA. OCTA in a single shot is able to obtain more information of the retina without the use of montage techniques. Despite the determination of retinal ischemia seems to be easier and more accurate using EFI SS-OCTA, FA offers more details of the perfusion status of the retina. In clinical practice the gold standard for the evaluation of retinal perfusion and neovascularization is fluorescein angiography (FA) . It is bThe development of optical coherence tomography angiography (OCTA) has provided a new noninvasive dyeless imaging technique to represent retinal vessels ,5. In coRecently, it has been described a new imaging technique for extending the scan length of OCT . A conveThe use of OCTA with an EFI technique may be used in the clinical practice to evaluate the retinal perfusion status in patients with vascular disorder. For this reason, the purpose of our study was to test the feasibility of swept source OCTA device, with and without the EFI technique and to compare this imaging technique to conventional FA.th 2017 to March 15th 2018 were prospectively included. Inclusion criteria were age ≥ 18 years old and the presence of either retinal or choroidal vascular disorders. Subjects with significant media opacities and poor cooperation were excluded from the study. All subjects underwent a complete ophthalmic examination, including a detailed medical history, refraction, intraocular pressure measurement, anterior segment and dilated fundus examination and FA performed using the 55-degree lens . The same day, OCTA imaging was performed using the PlexElite . An expert operator (MC) performed all the examinations using a 12 mm x 12 mm volume scan pattern centered on the fovea and using a + 20.00-diopter add-on prototype lens designed specifically by Zeiss to perform EFI examination and to avoid the positional instability of the lens. Following each examination, images were deemed acceptable if the retinal vessels were clearly visible and distinguishable.This study was approved by the Luigi Sacco Hospital Ethics Committee in Milan. A written informed consent was obtained from all subjects. The study followed the tenets of the Declaration of Helsinki for research involving human subjects. Consecutive patients with vascular disorder presenting to the Eye Clinic, Department of Biomedical and Clinical Sciences, Luigi Sacco Hospital, University of Milan from September 15The field of examination by EFI-OCTA was analyzed for each eye and compared with standard 55-degree field FA. Horizontal and vertical distances between two landmark points were measured using ImageJ software , as previously described –11. The Vessel density was calculated using ImageJ  ,12. In pConsequently, the binarized image was skeletonized and FD was calculated using Fractalyse  ,13. It iThe extension of retinal non perfused areas was evaluated by 2 graders (FC and MP) which measured manually and masquerade to the clinical background. Each image was displayed to the graders in casual distribution. FA images used for delineation of the extension of retinal non-perfusion were taken in the early phases, within 1 minute after dye injection, in order to reduce the effect of the leakage of the dye. Moreover, graders evaluated the presence and number of new blood vessels.Statistical analysis was performed using SPSS v.22.0 . All values were expressed as the mean ± standard deviation. Data were analyzed using Shapiro-Wilk test to evaluate the normality of sample distribution. The Wilcoxon signed rank test was used to compare the mean values between different groups. A P-value less than 0.05 was considered significant. Pearson’s rank correlation was used to determine the correlation coefficients of continuous variables. The agreement of measurements between readers was assessed using interclass correlation coefficient (ICC).A total of 43 eyes of 27 patients  were included in this study; 5 eyes of 4 patients were excluded due to image artefacts related to poor fixation. Thirty-two eyes (74%) were affected by diabetic retinopathy, 8 (19%) by retinal vein occlusion, 2 (5%) by retinal vein occlusion secondary to radiation retinopathy and 1 (2%) by Coat’s disease. Twenty-five eyes (58%) were treatment naïve whereas 18 (42%) underwent previous treatments including laser photocoagulation or with anti-vascular endothelial growth factor (VEGF) injections in the previous 6 months. The mean BCVA was 20/40   S1 FileS1 File.The mean extension ratio of EFI SS-OCTA compared to SS-OCT without EFI in the horizontal, vertical and overall direction were 1.40 ± 0.02 , 1.40 ± 0.01  and 1.97 ± 0.03 . The extension ratios in the two dimensions were not significantly different (P = 0.946).The mean extension ratio of FA compared to EFI SS-OCTA in the horizontal, vertical and overall direction were 0.92 ± 0.01 , 0.91 ± 0.01  and 0.85 ± 0.01 . The extension ratios in the two dimensions were not significantly different (P = 0.104).The mean extension ratio of FA compared to SS-OCTA without EFI in the horizontal, vertical and overall direction were 1.30 ± 0.01 , 1.29 ± 0.01  and 1.68 ± 0.02 . The extension ratios in the two dimensions were not significantly different (P = 0.122).2 , with SS-OCTA without EFI 20.42 ± 18.70 mm2  and with FA 27.56 ± 28.15 mm2 . The comparisons of mean extension of non-perfused areas between the different techniques were significantly different .The mean extension of non-perfused areas with EFI SS-OCTA was 34.24 ± 33.3 mmThe interobserver agreement of measurement of non-perfused areas between reader for EFI SS-OCTA was 0.879 (95% CI 0.846 to 0.953), for SS-OCTA without EFI 0.914 (95% CI 0.867 to 0.928) and for FA 0.892 (95% CI 0.871 to 0.933).The specificity and sensitivity for detection of new blood vessels were respectively 97% and 100% for EFI SS-OCTA 100% and 80% and for SS-OCTA without EFI. New blood vessels were found in 25.5% eyes with EFI SS-OCTA, in 18.6% eyes with SS-OCTA without EFI and 23.2% eyes with FA. No differences were found in term of mean number of new blood vessels between FA and EFI SS-OCTA . No differences were found about the mean number of new blood vessels between FA and SS-OCTA without EFI . No differences were found about the mean number of new blood vessels between EFI SS-OCTA and without EFI (P = 0.071). The interobserver agreement of detection of neovascularization between reader for EFI SS-OCTA was 0.905 (95% CI 0.862 to 0.948), for SS-OCTA without EFI 0.926 (95% CI 0.893 to 0.959) and for FA 0.967 (95% CI 0.935 to 0.987).The mean vessel density of EFI SS-OCTA was significantly different compared to SS-OCTA without EFI  P < 0.0001. The mean fractal dimension of EFI SS-OCT was significantly different compared to SS-OCTA without EFI  P < 0.0001.Using EFI SS-OCTA, VD was positively correlated to FD , the non-perfusion areas were negatively correlated to VD  and FD . Using SS-OCTA without EFI, VD was positively correlated to FD , the non-perfusion areas were negatively correlated to VD  and FD .OCTA is a recent imaging technique that allows qualitative and quantitative assessment of retinal and choroidal vessels in a limited area using computer-generated algorithms to determine blood flow ,5. One oIn this study we investigated the feasibility of EFI SS-OCTA in clinical practice by comparing its qualitative and quantitative parameters with standard FA and SS-OCTA without EFI. Our results documented that EFI SS-OCTA captured larger areas of the fundus than SS-OCTA without EFI and FA images with 55° field of view. This is a great advantage since EFI OCTA consists in a single fast acquisition able to obtain more informations of the retina without need for montage techniques that necessarily require extra time to obtain multiple scans, overlapping areas and an extra time to remove inaccuracies due to subtle misalignments.We also performed a qualitative and quantitative analysis regarding the presence and the number of new blood vessels finding no differences comparing several imaging modalities. In this contest, to evaluate new blood vessels is recommended the use of vitreoretinal interface slab that excludes the retinal vessels and enhances the visualization of new blood vessels protruding in the vitreous cavity as shown in On the other side, the evaluation of non-perfusion areas revealed a significantly larger extension with EFI SS-OCTA compared to FA and SS-OCTA without EFI. Consequently, the measurement of VD and FD revealed significant lower values with EFI SS-OCTA compared SS-OCTA without EFI. In fact, the extension of non-perfusion areas is negatively correlated with VD and FD, suggesting that the increase of non-perfusion areas is associated with a reduction of VD defined as the area occupied by vessels divided by the total area and consequently of FD. Moreover, VD is positively correlated with FD, suggesting that the increase of VD is associated with the increase of the branching complexity of the capillary network.Possible explanation of these results are related to the wider field of EFI SS-OCTA that could image additional areas of non-perfusion. In particular, we obtained in the same order wider imaged with EFI SS-OCTA, then FA and lastly with SS-OCTA and consequently larger areas of non-perfusion with EFI SS-OCTA, then FA and lastly with SS-OCTA. At the same time, we have to consider that the use of EFI technique can potentially overestimate the non-perfusion areas due to an over-representation of the edges of the images where the curved surface is projected flat.Another factor that should be taken into account is the different resolution of the images obtained. In fact, EFI SS-OCTA cover larger area then SS-OCTA without EFI with the same number of A-scan and resulting in a lower resolution of the image . This diOther factors that could influence the analysis are blood flow velocity and possible errors in the segmentation boundaries. Particularly slow flow may result in black signal misinterpreted as non-perfusion in OCTA and not corresponding to FA . HoweverOur results are in agreement with previous studies that investigated the EFI SS-OCTA with standard FA ,11. HiraDifferently, Kakihara et al.  evaluateThe present study has several limitations as the small number of included eyes. Moreover, we have to consider that other factors may have influenced the results of our analysis as the poor pupil dilation or media opacities. At least, in our series we included a limited subset of pathologies and with only a retinal involvement.In conclusion, we found that a single capture of EFI SS-OCTA covers a larger area of the retinal vasculature than conventional FA. Moreover, the use of EFI SS-OCTA in the clinical practice can save a lot of time, as several OCTA images have to be made for one montage and may burden the cooperation of the patient. Despite, the determination of retinal ischemia seems to be easier and more accurate using EFI SS-OCTA, FA offers more details of the perfusion status of the retina as it is not influenced by the velocity of the flow. At this moment, FA should be considered as the gold standard for the evaluation of retinal perfusion. Furthers studies are needed on the comparison between FA and OCTA to clearly assess the condition of diseases affecting the retinal vasculature in more peripheral regions.S1 File(XLSX)Click here for additional data file.


A static model was constructed with ex vivo porcine corneas clamped in Franz cells. To test the predictive capacity of models, four different eye drops containing sodium hyaluronate were spiked with tritium-labeled sodium hyaluronate to standardize quantification. In both settings, eye drops were applied for 5 min and physiological conditions were mimicked by flushing with artificial tear fluid. Spreading experiments on HCE-T next to synthetic membranes were used for further characterization.Two different, complementary corneal binding set-ups were developed. In a dynamic i.e., to prevent calcification) were used, binding was not influenced. All eye drops spread immediately when placed on HCE-T and at the same order of magnitude on glass and polyethylene terephthalate surfaces.Binding was more pronounced in dynamic HCE-T model. Three of the four eye drops demonstrated sigmoidal elution of sodium hyaluronate, suggesting pronounced binding. One solution eluted distinctly faster, likewise the buffer control. The static method produced a similar ranking but at lower levels. When eye drops in which phosphate buffer was replaced by citrate buffer (in vitro to corneal surfaces.Dynamic and static models performed on different corneal sources were used to determine sodium hyaluronate binding kinetics in solutions under physiological conditions. These methodologies resulted in a ranking of the capacity of sodium hyaluronate to bind  Additionally, most molecular weights of the hyaluronates that are incorporated into eye drops vary or are unknown.Dry Eye Disease (DED) is a common condition around the world that affects up to one in every three people . Althougin vitro studies was to develop methodologies that can be used to assess the magnitude and binding kinetics of Na-HA in eye drops used on corneal surfaces and to compare adhesion among the respective eye drops in two different settings. Representative commercial eye drops with different compositions were selected and design-supplemented by buffer control. All of the selected eye drops contained Na-HA but in different amounts. The amounts ranged from 0.10% Na-HA in HYLO-COMOD® and Hyaluron-ratiopharm® eye drops to 0.15% in Bepanthen® eye drops and BLUpan® medical UD. To standardize and allocate in vitro binding by sodium hyaluronate, the methods were designed to take into account elaborated corneal surfaces, including monolayers of cultured Human Corneal Epithelial Cells (HCE-T) and ex vivo porcine corneas. Both in vitro corneal settings have been characterized in detail regarding their corneal permeation  sodium salt (MW 300.000 Da) with a specific activity (assay January 2016) of 260 mCi/g in sterile water was obtained from American Radiolabeled Chemicals Inc., USA. Material was stored at recommended temperature of 0-5°C and used within 6 months. Each spiking was performed immediately before start of the experiments.2.1.3The tear fluid used in this study was composed of 0.68% sodium chloride, 0.05% potassium chloride, 0.014% sodium dihydrogen phosphate monohydrate, 0.21% sodium bicarbonate, 0.36% HEPES, 0.11% D-Glucose monohydrate, 0.02% magnesium sulfate heptahydrate, 0.01% calcium chloride dihydrate and lysozyme extracted from human milk powder (1.2 g/L). All substances (except lysozyme) were dissolved in double-distilled water. After the solution was equilibrated to room temperature, lysozyme was added, and the pH was adjusted to 7.4 with NaOH or HCl. The artificial tear fluid (ATF) was then sterile-filtered and stored at -20°C until used.2.1.4in vitro adhesion was performed using an ex vivo model of porcine corneas and FC. Porcine eyes were obtained from a local slaughterhouse and stored in sterile PBS until used. The corneas were excised and immediately mounted in FC (incubation area per cornea: approx. 0.64 cm2).An assessment of 2.1.5In vitro adhesion studies were performed with HCE-T monolayers on Chamber Slides (CS). The dimensions were 50 mm long and 18 mm wide (including a 9 cm2 growth area for the monolayer). The following were the total dimensions of the CS: 76 mm x 24 mm . For details, see Fig. (1).Cell monolayers composed of human corneal epithelial cells were used. SV40-transfected Human Corneal Epithelial Cells (HCE-T cells)  were cul2.22.2.1in vitro adhesion via scintillation counting. All products were used unaltered, and all flasks were opened immediately before they were spiked with 3H-Na-HA for in vitro tests. The final levels of radioactivity observed in the FC experiments were 0.5 µCi/mL and 1.0 µCi/mL performed in the declining Channel (dC) set-up.As no information is currently available regarding the molecular weights of the Na-HA in each of the different products used in this study, tritium-labeled Na-HA (3H-Na-HA) was incorporated into the eye drops to facilitate a direct comparison of the effects of the formulations on adhesion. Moreover, an assessment of 3H labeling allowed us to perform an analytical quantification of 2.2.2n = 3).Immediately after the corneas were clamped into FC, the surfaces were incubated with 3H-Na-HA eye drops or PBS , and the corneas were incubated for 5 min while gently rotating at 100 rpm. A stepwise dilution with ATF was then performed for 10 min as follows. Every minute, 200 μl of a donor solution was replaced with an equal amount of ATF. The amount of 3H-Na-HA in the donor solution was determined both before and after incubation in addition to after dilution. The amount of 3H-Na-HA on the corneal surfaces was determined after the experiments ended. All studies were conducted in triplicate . This blue dye allowed the direct visualization of flow characteristics. The flow rate was initially started at 0.6 to 0.8 mL/min and was decreased to a 33- to 44-fold lower rate of 18 µL/min. Finally, it was necessary to use two needles to avoid the formation of “undiluted” zones at the edges of the CS. CS with HCE-T monolayers were placed on dC at an angle of approximately 11°. The angle was determined based on the height of a petri dish (15 mm) and the total length of the chamber slide (76 mm). A defined volume of 35 µL of 3H-Na-HA-spiked eye drops or PBS was applied to the upper end of the CS (between the two needles) containing HCE-T monolayers. The 35 µL volume (corresponding to approximately one ED drop) was applied while the CS was already at 11° and 250 µL buffer was applied at the bottom. In accordance with the study design used in the FC/ex vivo porcine cornea experiments, the duration of exposure was fixed at 5 min. All studies were conducted six times (n = 6).In the dynamic study design, we used the dC model and  tissues  and was  tissues , 19. Theout rate , 21. In 2.2.4in vitro tests were performed (each experiment was performed six times).The process of spreading the cells on the corneal surfaces prior to the experiments was uncoupled from the static and dynamic corneal evaluations and investigated by measuring the contact angles of the different surfaces. The dynamic contact angles of the eye drops were measured on membranes using a G10 contact angle analyzer  equipped with an axisymmetric drop shape analysis profile. The surfaces exhibited varying physical and biological properties. Glass (hydrophilic) and polyethylene terephthalate  had solely physical characteristics. Cell monolayers of HCE-T cells were grown on chamber slides and used to evaluate biopharmaceutical relevance. The eye drops were equilibrated to room temperature at least 24 h before these experiments were performed. During measurement, whole-drop geometry was recorded and mathematically fitted. Fitting of the contact angles was determined for solid-to-liquid boundary surfaces. All eye drops used in the determinations were used unaltered and were immediately opened before 2.2.5In vitro samples (200 µL FC or 100 μl dC) were transferred into 2000 µL scintillation cocktails and measured on an LS-6500 Liquid Scintillation Counter .2.2.6k*(k-1)/2 pairwise comparisons were performed in which k represented the number of the solution being investigated. The results were tested using Dunn’s test. All p-values were adjusted using the Bonferroni correction. Only corrected p values less than 0.05 were considered statistically significant. Computations were carried out using R and the PMCMR package.The data are shown as the mean ± Standard Deviation (SD). For each experimental setting, 33.1ex vivo porcine corneas . With regard to the amount of 3H-Na-HA that was applied at the start of the experiment, all of the tested eye drop formulations had comparable retention rates, as follows: 0.23% ± 0.03% for HYLO-COMOD®, 0.10% ± 0.02% for Hyaluron-ratiopharm® eye drops, 0.12% ± 0.05% for Bepanthen® eye drops, and 0.13% ± 0.04% for BLUpan® medical UD. In comparison, a considerably lower amount of 3H-Na-HA was obtained from the PBS-treated corneas (0.07% ± 0.01%). This amount was close to the quantification limit of the scintillation method. The total mass balance after the experiment was in the range of 81-102%, indicating a complete recovery. Although the data plots appeared to indicate that there were differences between the solutions, only HYLO-COMOD® was found to be significantly different from PBS (padj ≈ 0.026).In the FC setting, only a small amount of 3H-Na-HA (close to the quantification limit) adhered to the 3.2®, 1.4% ± 0.3% for Hyaluron-ratiopharm® eye drops, 6.5% ± 3.8% for Bepanthen® eye drops and 3.8% ± 1.3% for BLUpan® medical UD. This order was similar to that observed for 3H-Na-HA binding by the respective eye drops. As in the FC setting, the amount of 3H-Na-HA that was obtained from PBS (0.9% ± 0.1%) was considerably lower . The total mass balance after the experiment was in the range of 87-110%, indicating a complete recovery. A statistical comparison of the Bepanthen® eye drops, the BLUpan® medical UD (padj = 1) and HYLO-COMOD® (padj = 1) showed that there were no differences among these treatments. However, there was a significant difference between the Hyaluron-ratiopharm® eye drops (padj ≈ 0.032) and the PBS control (padj < 0.001).Consistent with the FC and porcine cornea experiments, the results of the declining channel set-up of HCE-T monolayers grown on CS showed that the retention rates were 3.8% ± 1.4% for HYLO-COMOD4) of 3H-Na-HA revealed that there were differences between the tested products. While HYLO-COMOD®, Bepanthen® eye drops and BLUpan® medical UD exhibited a sigmoidal increase in 3H-Na-HA over time, Hyaluron-ratiopharm® eye drops eluted distinctly faster in a pattern similar to that of PBS.An analysis of the elution kinetics  and BLUpan® medical UD (citrate buffer), no significant difference was confirmed .With regard to the difference in buffer between Bepanthen3.31) measured on hydrophobic PET surfaces were pronounced, whereas the angles on HCE-T monolayers were negligible and allowed complete spreading. Spreading on glass surface was in-between HCE-T and PET. For all products, the mean contact angles on glass surfaces ranged from 42.9° to 48.9°, while those on PET surfaces ranged from 105.7° to 112.1°. In each subgroup of glass and PET surfaces, the contact angles were within the same order of magnitude. A statistical analysis of Bepanthen® eye drops versus BLUpan® medical UD showed that there was no difference in the contact angles they produced on glass (padj = 1) or PET surfaces (padj = 1), indicating that the switch from phosphate buffer in Bepanthen® eye drops to citrate buffer in BLUpan® medical UD had no impact on spreading. While no statistically significant differences were found for glass, spreading on PET was significantly different between the Hyaluron-ratiopharm® eye drops and the Bepanthen® eye drops (padj ≈ 0.002) and BLUpan® medical UD (padj ≈ 0.017). On the biological surface , all of the eye drops spread over the surface immediately after the drop hit the surface . Adjusted p-values for all pairwise comparisons between contact angles for glass and PET surface were summarized (Table 2).The contact angles  and severity gradings. Tear Film and Ocular Surface Society (TFOS) updated an evidence-based definition and a contemporary classification system for DED [A comprehensive three-part classification framework of DED summarized , 23 the  for DED , which c for DED . Pathoph for DED , 27 desc for DED , 29. An  for DED  included for DED  and the  for DED .via proteomics and metabolomics of tear film [The comprehension of normal tear film  and impoear film , 35, bioear film -38 or seear film . Changesear film  accentuaear film . Three kvia nasolacrimal duct. The outermost third part of the tear film is a thin layer of lipids. This lipid layer reduces the surface tension of film and re-spreading after blinking. Lipid movement and film reconstruction is relatively fast and driven via a concentration gradient of polar lipids. Within tear film break-up, the film thickness will reduce until collapse. In humans, time is in-between seconds and up to one minute. In DED period might be further reduced to few seconds or the breakup can even occur instantaneously after a blink [The mucin layer (first layer) is directly located at the corneal surface and anchored to the epithelium. Epithelial cells produce the main component of sugar-rich glycosylated proteins and form a gel-like structure. This easily wettable surface layer assist in water re-spreading after blinks . Differe a blink . Meibomi a blink . Accordi a blink , 49 and  a blink .® and Bepanthen® eye drops were assigned to category three, Hyaluron-ratiopharm® and BLUpan® medical UD were unfortunately not included in this survey. These rheological groups were in agreement with the outcomes of the present in vitro corneal binding experiments.To overcome an impaired fluid layer, Na-HA is ideally suited because it displays a long-lasting, intensive ocular lubricant . Additioin vitro and in vivo. In many cases, the depth of application of Na-HA was the focus of these studies. Chronologically, the first investigations used gamma scintigraphy [in vitro findings, the CRT of Na-HA was rather short (approximately 3-10 min) but superior to that of the PBS control. Additionally, Na-HA CRT was not different between healthy and keratoconjunctiva sicca groups, and increasing Na-HA concentrations led to increased binding. Finally, the impact of 0.2% Na-HA on CRT was superior to that of 0.3% HPMC and 1.4% PVA [i.e., via HPLC. Dependencies that affect CRT have mostly been studied in rabbits, in which different polymers, including Na-HA [in vivo method that has been used in rabbits and humans involves the use of fluorimetry measurements to determine the precorneal residence time of novel formulations containing non-penetrating FITC dextran (MW 70-73 kDa) [50 values to be determined. In another study, fluorescein sodium dissolved in 0.1% HA solution and 0.1% fluorescein-conjugated with hyaluronic acid (FHA) dissolved in saline were instilled into the eyes of healthy volunteers [in vitro binding experiments aimed at exploring Na-HA CRT are in line with and showed similar periods to FHA studies and showed that Na-HA was superior to the buffer controls.A total of three major approaches have been implemented during the years that allow Corneal Residence Times (CRT) to be determined and visualized tigraphy -57 with tigraphy -55. Alte1.4% PVA . A secon1.4% PVA -60, i.e.ng Na-HA , nanoparng Na-HA  and mixtng Na-HA -65. An i-73 kDa) . This telunteers . After 1lunteers , mucoadhlunteers  and actiin vitro binding experiments confirmed the results of in vivo experiments, performed using currently available techniques, with regard to the grading and extent of Na-HA binding that was achieved. In general, the use of tritium-labeled Na-HA allowed us to combine the sensitivity of gamma scintigraphy and fluorescence assays with the direct detection of Na-HA. This was similarly true for the tear fluid assays performed without structural drug modifications. Hence, our simplified method produces results faster than later in vivo studies and allows the ability to establish predictiveness and selected physiological conditions and to make in vivo correlations. Additionally, this method can be quickly implemented. A retrospective inspection of the Na-HA CRT data illustrated further validation parameters relevant to CRT, such as different drug delivery strategies, and their effects on Na-HA. These include adhesive polymers, nanoscale formulations and dose-response curves for Na-HA. Moreover, our in vitro binding assays avoid the time spent during the early stages of development in addition to avoiding the cost-consuming animal and human trials. The clinical data on keratoconjunctiva sicca patients [in vitro CRT binding in an in vitro disease model might be a future optimization step. Finally, the combination of permeability assays and CRT binding was introduced for use with HCE-T monolayers [The results of patients , 55 shownolayers .in vitro model was used, the binding behaviors of Na-HA in HYLO-COMOD® and Bepanthen® eye drops were comparable. Furthermore, their binding kinetics had similar profiles. We used in vitro models (static versus dynamic) independently to assess the adhesion rates and binding kinetics of Na-HA on the ocular surface under physiological conditions by controlling the amount of eye drop fluids that were applied or how much tear fluid volume exchange occurred. These analyses showed that similar results were obtained with regard to how the formulations were ranked in both in vitro settings. Binding effects were more pronounced in the HCE-T model. In both set-ups, Na-HA binding was the lowest in the control PBS formulation. In the HCE-T model, the elution kinetics of Na-HA were different between the tested eye drops. While most of the eye drops showed a sigmoidal increase in Na-HA, suggesting pronounced binding, Hyaluron-ratiopharm® eye drops exhibited a pattern similar to the elution profile of the PBS control. In addition, the HCE-T model, when used in the declining channel set-up, provided the first simple evidence that allowed us to evaluate reformulations of eye drops. Over the years, changes in buffer media have likewise occurred, such as in Bepanthen® eye drops (phosphate buffer) and BLUpan® medical UD (citrate buffer), without any further modifications. Biological responses were evaluated by spreading experiments performed on synthetic surfaces. Clinical observations of corneal calcification that was attributed to the presence of phosphate within eye drops  have been characterized [ex vivo Eye Irritation Tests (EVEITs). Previous clinical observations showed that the topical use of artificial tears containing phosphate in injured eyes led to sight-threatening corneal complications in high-risk patients. By simulating these treatment conditions, the EVEIT convincingly demonstrated that changes in the composition of pharmaceutically administered treatments, similar to switches in the buffer from phosphate to citrate buffer, can prevent this undesired side effect. Eye drops containing citrate buffer did not cause corneal calcification in the eyes.In our studies, regardless of whether a static or dynamic cterized  in ex viin vitro setting should be mentioned. Changes in parameters might be driven by natural circumstances in the in vitro models or the experimental set-up conditions. On the one hand, differences might be due to differences in the in vitro structure of the corneal surface used in HCE-T and that observed in ex vivo pig corneas. Although they were highly correlated [ex vivo corneas. Second, differences in incubation times, periods and handling  can affect results. Because the binding capacity of Na-HA is low, these parameters, although kept constant within the assays, might have impacted the results. The most moderate set-up, the HCE-T monolayer, when used with a flow rate of 18 µL/min, produced higher bindings. A third parameter, the proportion of the in vitro model surfaces and the applied volumes, was also different across the conditions. For example, 400 µL of 3H-Na-HA-spiked eye drops or PBS were applied to a 0.64 cm2 surface in the FC and pig corneas, whereas in the HCE-T monolayer set-up, 35 µL was applied to a surface area of 9 cm2 (50 mm x 18 mm). In the declining channel method, only approximately 80% of the monolayer could interact with this volume, so that the experimental area was decreased to 7.2 cm2. In the FC experiments, 1 cm2 was exposed to a volume of 625 µL, whereas in the HCE-T model, 1 cm2 was exposed to a volume of 5 µL. Consequently, there were differences in the areas of corneal surfaces that could bind the formulations.Several parameters and their combined impact on binding in the different rrelated , the HCEin vitro binding methods are valuable supplements to well-known physical tests of spreading and rheology. Additionally, the data obtained can be linked to the interplay with the corneal surface to fill in the gaps in the cornea in in vitro permeability tests [In vitro corneal retention studies provide techniques that can provide further support within early formulation development and to classify mucoadhesive polymers [Our novel ty tests . In vitrpolymers , their min vitro methodologies produced comparable evidence and rankings based on the determination of the rate of Na-HA binding to ocular surfaces in eye drops. In most cases, eye drop solutions enabled a higher extent of Na-HA binding than was observed in the PBS control. In general, the bindings were weak and might be more precisely described as retention. Furthermore, the HCE-T monolayer model provided a functional in vitro approach for determining the binding kinetics of Na-HA. Moreover, this method allows researchers to mimic lacrimation by using physiological levels of ATF.Both in vitro studies enhance our knowledge about the capacity of Na-HA in eye drops to bind to the ocular surface. However, the in vivo relevance of these results should not be over-estimated. In fact, the in vitro designs depicted here represent important steps that support the development of eye drops that are to be applied to the corneal surface. However, their clinical significance is limited due to, for example, missing eyelid movements, varying dilutions on the surface and the lack of lacrimation. The HCE-T in vitro kinetics model might be a useful tool for studying the effects of formulations on corneal binding during drug development. Likewise, as was demonstrated for Bepanthen® eye drops, which contain a phosphate buffer, versus BLUpan® medical UD, which contains a citrate buffer, the in vitro binding assays demonstrated the potential to test the extent of equivalence with regard to binding kinetics. Finally, spreading experiments performed on glass and polyethylene terephthalate demonstrated the power of these tests to explore non-biological effects.The results of our in vitro set-ups could be used as valuable discriminators to assess the effects of, for example, changes in preservatives or in the classification of new excipients with adhesive properties. In the future, it would be of interest to further correlate in vivo binding results to optimize experimental designs.Within the development of locally acting eye formulations, a novel


Epinephelus coioides) has been shown to have extensive potentials as an alternative for current antibiotics. Due to the huge costs for the study and the production of a new drug, if an antimicrobial peptide has other beneficial functions in addition to antimicrobial activities, it would be preferred.Antibiotic-resistant pathogens are an emerging threat in this century. Epinecidin-1 is a multi-functional Antimicrobial Peptide (AMP) produced by Orange-spotted grouper (In this study, properties and applications of Epinecidin-1 were investigated and addressed comprehensively. To achieve this, the Google Scholar search engine and three databases of PubMed, Scopus, and Web of Science were used.Epinecidin-1 is a cationic AMP with an alpha-helical structure. Seven functional usages of this peptide have been reported in the literature including antibacterial, antifungal, antiviral, antiprotozoal, anticancer, immunomodulatory, and wound healing properties. Moreover, this peptide has high potential to be used as an active ingredient in cleaning solutions as well as application in vaccine production.Staphylococcus aureus and Helicobacter pylori and also wound healing properties, Epi-1 has high potential to be considered as an important candidate for the production of new drugs and treatment of various infections including diabetic foot ulcer and peptic ulcer. Moreover, adjuvant-like properties of Epi-1 make it a suitable candidate for the studies related to an adjuvant. Other attractive properties such as anticancer effects have also been reported for this peptide which encourages further studies on this peptide.Due to significant antimicrobial activities tested on bacteria such as  The emergence of antibiotic-resistant pathogens is one of the main concerns of health organizations in recent years. Various types of drug resistance are rapidly being developed for the pathogens while were previously treatable using simple antibiotics. However, the introduction process of new antibiotics is greatly slow. Evidence shows that the risk of post-antibiotic era is relatively close to humans , 2. TherAntimicrobial Peptides (AMPs) are one of the most potent alternatives, and many studies have been conducted for them in recent years. They usually have a short length (less than 50 amino acids), and cell membrane attack is the most known mechanism of their antimicrobial activity. AMPs are often extracted from a living organism, or they are peptide derivatives . With thEpinephelus coioides)  Fig.  has beenountries .Fig. 1OrUp to now, many studies have been performed to identify the Epinecidin-1 and mechanism of action. In this review, all studies related to this AMP are investigated with the aim of introducing various classified aspects of this peptide.The search was performed in two steps. First, all English studies including the Epinecidin-1 AMP  were selected from PubMed, Scopus, Google Scholar, and Web of science databases. Only those original studies were included in our study that helped to clarify one of the Epinecidin-1 aspects, at least in one part or the whole of their research.The search formula (Epinecidin-1[All Fields] OR Epinecidin-1[All Fields]) AND antimicrobial peptide[All Fields] was used for PubMed. The search formulas for Scopus and Web of science were similar to the PubMed search formula. The keywords of Epinecidin-1, Epi-1, Epinecidin-1 antimicrobial peptide and Epi-1 antimicrobial peptide were used for the Google Scholar search. In addition, the cited studies to our selected articles and references were reviewed and studies that could provide possible additional information on the structure, mechanism, and source of Epinecidin-1 were selected.E. coioides. cDNA of Epinecidin-1 has 518 bp length (GenBank: AY294407.1), and its largest open reading frame is 204 bp. Epi-1 is initially synthesized as a 67 amino acids prepropeptide containing three domains of the signal peptide, mature peptide, and a prodomain. The Epi-1 gene consisted of three introns and four exons. Determination of domains and the number of amino acids have been performed by bioinformatics software, as the first 22 amino acids were considered as a signal peptide and the next 25 amino acids as the mature peptide [E. coioides showed that the most expression of this AMP was related to the head, kidneys, intestines, and skin. While the immunohistochemical analysis showed that the peptide was localized in the gills and intestines [Vibrio harveyi-infected Grouper has shown that expression of this peptide upregulated by the onset of infection and persisted for ten days. Interestingly, the highest expression occurred in the skin followed by gills, liver, and kidney [Yin et al. in a study in 2003 made an orange-spotted grouper leukocyte cDNA library and sequenced more than 300 expressed sequence tags (ESTs) of cDNA . Three ytestines . This cad kidney .Table 1EE. coioides is increased in exposure to the Lipopolysaccharide (LPS) and Polyinosinic:polycytidylic acid (poly(I):poly(C)) in a dose-dependent manner [Investigation of 1182 bp upstream of the Epinecidin-1 gene showed that there were some putative regulatory elements and binding motifs for transcription factors in this region. These sequences included TATA box (nucleotide − 34), three binding sites for CAAT enhancer-binding protein β (C/EBPβ) , and two binding sites for hepatocyte nuclear factor (HNF) 1 (nucleotide − 667) and HNF-3b (nucleotide − 951). It has also shown that the Epinecidin-1 production in t manner , 12.23-47 with other AMPs isolated from fish has shown that this peptide has a 71.4–78.6% identity with three peptides of Chrysophsin 1, 2, 3. After chrysophsins, the most identity was related to Moronecidin (69%), Pleurocidin WF3 (61.9%), and Piscidin3 (54.5%), respectively [Comparison of Epinecidin-1ectively , 15.Three derivatives of Epinecidin-1 have a molecular weight of 2.3–2.9 kDa and are highly cationic  was 39 μg/ml compared to the 9.7 μg/ml of the unirradiated control after exposing to methicillin-resistant Staphylococcus aureus (MRSA). Temperature rise also reduced the antimicrobial activity. The MIC was 312 μg/ml in exposure to MRSA after 5-min pretreatment at 40 °C compared to the control group which was 9.7 μg/ml at 25 °C. While temperature and irradiation showed a negative correlation to antimicrobial activity, acidic conditions had no significant effect on its activity. The highest activity of Epinecidin-1 was found under acidic conditions [The stability of Epinecidin-1nditions .AMPs usually have extended-spectrum effects . So far,Riemerella anatipestifer infection, a study was carried out in 2009 [R. anatipestifer infected ducks as pretreatment, co-treatment, and post-treatment showed that the number of bacteria in duck greatly reduced in all three cases and the survival rate was significantly increased [Oreochromis mossambicus) and grouper to reveal the therapeutic effect of Epinecidin-1 in Vibrio vulnificus infection showed that the simultaneous injection of bacteria and peptide significantly increased their survival. The half-life of this peptide in tilapia was 60–80 min following the injection [V. vulnificus contaminated fish using zebrafish model system, it was shown that the pretreatment, co-treatment, and post-treatment with 1 μg of Epinecidin-1 could increase the survival rate of fish from 3 to 56.6%, 80, and 60% respectively [To evaluate the therapeutic effect of Epinecidin-1 on  in 2009 , 22. Invnjection . In a stectively .Pseudomonas aeruginosa. The mouse model infected by multidrug-resistant P. aeruginosa strains also combated the infection more effectively with no adverse behavioral effects, hepatotoxicity, and nephrotoxicity when Epinecidin-1 applied [Epinecidin-1 showed good bactericidal effects when exposed to sensitive and antibiotic-resistant  applied , 25.Helicobacter pylori is a gram-negative bacterium that causes active chronic gastritis and may cause complications such as malignancies, dyspepsia, and peptic ulcer [H. pylori infection is very important as the Nobel Prize in physiology in 2005 was awarded for the discovery of the peptic ulcer disease caused by an infection with H. pylori [H. pylori strains, evaluation of AMPs has started for the treatment of this infection. Epinecidin-1 is one of the most potent AMPs which have been introduced so far [H. pylori strains with the MIC of 8–12 μg/ml. It has also shown that the oral administration of Epinecidin-1 (250 μg) could completely remove the H. pylori from the stomach in a C3H/HeN mice [ic ulcer . The H. . pylori . FollowiHeN mice .Trichomonas vaginalis. More than 140 million people worldwide are annually affected by this infection [T. vaginalis, respectively [In a study performed in 2019 on mice, suffering from metronidazole-resistant trichomoniasis, it was reported that Epinecidin-1 could combat the infection effectively . Trichomnfection . In two ectively , 31. It ectively .Antiviral effects of many AMPs have been reported so far. However, according to the Antimicrobial Peptide Database (APD), antiviral effects have been evaluated and reported for only 185 out of more than 3000 registered AMPs. It should be noted that regarding the more difficult evaluation of antiviral properties, most of the AMPs have not been evaluated for these effects. Fortunately, Epinecidin-1 is among the AMPs with well investigated antiviral properties .E. coioides infected by NNV and the best result was related to the co-treatment. Co-treatment of Epinecidin-1 and NNV through the anal canal could increase the survival rate of infected fish in a dose-dependent manner. For example, co-treatment of 1 μg/fish Epinecidin-1 ten minutes before the virus inoculation could increase the survival rate from 11 to 35% [Betanodavirus or nervous necrosis virus (NNV) is a virus classified in the Nodaviridae family. It is one of the main fish pathogens which causes much damage in cultured marine fishes each year . Effect 1 to 35% . In anot1 to 35% .22-42 could combat FMVD more effectively. The BHK-21 cells were used in their study. The 50% cytotoxic concentration (CC50) of Epinecidin-1 for BHK-21 cells was 19.5 μg/ml and the 50% effective concentration (EC50) for viral inhibition was 0.6 μg/ml. Investigation of higher concentrations (10 × EC90 of Epinecidin-1) showed good virucidal activity against FMDV. It was also observed that the most effect of this peptide was during the viral adsorption stage [The foot-and-mouth disease virus (FMDV) is a member of the picornavirus family and Aphthovirus genus that can cause foot-and-mouth disease in cloven-hoofed animals and is highly contagious. Although it has a low mortality rate, it can cause significant losses for animal husbandry in low milk and meat production as well as animal products . In a ston stage .Japanese encephalitis virus (JEV) is a flavivirus that causes encephalitic diseases in children . It has E. coioides [Evaluation of the gene expression of Epinecidin-1 under different conditions showed that the infection by Singapore grouper iridovirus (SGIV) up-regulated the Epinecidin-1 gene in coioides .Most of the current cancer treatment methods are complex and have side effects. For example, chemotherapy causes serious side effects. Also, current anticancer drugs are often focused on the destruction of cells with a high rate of growth, and probably they are not able to differentiate between cancer and normal cells. In addition, the emergence of multidrug-resistant cancer cells which are capable of neutralizing the function of anticancer drugs is a new challenge. This happens through various mechanisms including repair of damaged DNA, transport of the drug out of the cell, and expression of drug detoxifying enzymes , 42. ConAnticancer AMPs are usually divided into two groups. The first group can kill cancer cells as well as normal mammalian cells. The second group is potent to kill cancer cells, while no activity against normal mammalian cells or with poor activity . A studyIn another study, it was shown that Epinecidin-1 inhibited the proliferation and induced the apoptosis in human leukemia U937 cells. This seemed to occur through the increase in tumor necrosis factor (TNF)-α; interleukin (IL)-10, interferon (IFN)-γ, p53, IL-15, and IL-6. It was also observed that the ADP/ATP ratio which is an indication of mitochondrial dysfunction, was increased in these cells .To date, there is not much research to clarify the exact mechanism of Epinecidin-1 anticancer function. However, it should be noted that the AMPs with a similar structure to Epinecidin-1 usually have the same anticancer mechanism . GeneralEpithelial cell proliferation and migration are two important factors in wound healing . The effFurthermore, animal studies on MRSA-infected skin ulcers in mice and swine indicated that this peptide reduced the bacterial count in the wounded region, increased the angiogenesis, and enhanced wound closure , 50.In a mouse model with MRSA-infected skin ulcer, use of Epinecidin-1 resulted in faster wound closure compared to the non-infected ulcers and infected but vancomycin-treated ulcers. Also, co-treatment with Epinecidin-1 and collagen enhanced the healing and re-epithelialization compared to Epinecidin-1 treatment alone. Evidence shows that Epinecidin-1 activity in MRSA-infected ulcers is not only related to the antimicrobial property, and this peptide also plays a role in the regulation of some cytokines. For example in MRSA-infected mice ulcers, treatment with Epinecidin-1 decreased serum levels of the proinflammatory cytokines TNF-α, IL-6, and MCP-1, and regulated the recruitment of monocytes and clearance of lymphocytes around the wounded region during healing .In confirmation of previous findings, the results of another study on the effect of Epinecidin-1 on the healing of MRSA-infected swine burn ulcers showed that this peptide could accelerate the wound healing and provide protection against infection. Within 25 days, Epinecidin-1 increased epithelial activities, extracellular collagen compound formation, and vascularization. Also, treatment of swine with Epinecidin-1 suppressed the C-reactive protein (CRP) and the pro-inflammatory cytokine IL-6, while their expression induced in untreated swine group .In this section, the discovered mechanisms of action of this peptide are introduced. In most studies, there are two main mechanisms involved to combat the infections, including (i) Direct attack on the cell membrane of pathogens (ii) Regulation of the immune system to combat infection.Candida albicans, Propionibacterium acnes, and Trichomonas vaginalis in exposure to Epinecidin-1 were evaluated in one study using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The comparison of their appearance before and after Epinecidin-1 treatment showed that this AMP caused breakage in the membrane and intracellular contents were transported outside the cell. C. albicans was also found to have a highly irregular shape, in addition to the membrane breaks. The morphological changes of Trichomonas were as membrane destruction and hollow appearance [R. anatipestifer which is a pathogen of ducks, and it was shown that this peptide caused breaks in the membrane and the efflux of cellular contents [P. aeruginosa using TEM revealed membrane disruption and lighter electron density in the cytoplasm [The main invasive mechanism of this peptide in exposure to pathogens is the destruction of the cell membrane , 31. It contents . Furtherytoplasm .Fortunately, the toxic concentration threshold for human cells is much higher than the microbial cells. Epinecidin-1 at the concentration of 0.42 μM have no hemolytic effect on human RBCs. However, hemolytic effects appear to be highly dose-dependent at the 0.83 μM concentration , 29. HigIt has been shown in many studies that Epinecidin-1 combat infections through the regulation of the immune system. In Japanese encephalitis virus infection, Epinecidin-1 modulated the expressions of MCP-1, IL-6, IL-10, IFN-γ, TNF-α, and IL-12, and elevated the levels of anti-JEV-neutralizing antibodies in the serum .V. vulnificus-infected fish, Epinecidin-1 could also regulate immune response genes such as IL-10, IL-1b, TNF-α, and IFN-γ and help to control the infection by enhancing the immunity [It was also shown that Epinecidin-1 could play a role in the regulation of Mx2 and Mx3 genes expression , 35. It immunity .P. aeruginosa in a mouse model showed that this peptide was a good immunomodulator. Epinecidin-1 induced significant secretion of immunoglobulin G1 (IgG1) through the activation of Th2 cell response in infected mice. Treatment with this peptide during the septic shock also had a positive effect and probably involved protecting the liver against LPS-induced dysfunction and preventing damage to several other organs in septic mice [Co-injection of Epinecidin-1 and tic mice .E. coioides) and zebrafish (Danio rerio), it was shown that this peptide significantly increased the expression of some important genes of immunity such as (TNF)-1 in grouper and Toll-like receptor (TLR)4, IL-1b, nitric oxide synthase (NOS)2, and (NF)-κB in zebrafish [In a study of immunostimulatory effect of Epinecidin-1 peptide in two models of grouper  to express Epinecidin-1. It was observed that supplementing adult or larva fish especially zebrafish with this transgenic Artemia strain, significantly increased the immunity against infections , 54.P. aeruginosa ATCC 19660, it was shown that Epinecidin-1 decreased the concentrations of IL-1β, IL-6, and TNF-α. Interestingly, if the peritonitis caused by the resistant strains of P. aeruginosa, Epinecidin-1 treatment significantly increased plasma concentrations of IL-1β and TNF-α [In the study of mice peritonitis infection caused by nd TNF-α . Epinecind TNF-α , 56.H. pylori infection through immunologic changes in the host. For example, inhibited infection through in vivo depletion of CD4 + -FOXP3+ T Regulatory and Th17 subset populations, and aided in clearance of persistent H. pylori colonization. Flow cytometry and gene expression analysis of mouse splenic and gastric tissue indicated that Epinecidin-1 inhibits IL-10, and thereby affects FOXP3 expression levels and reduces pro-inflammatory cytokine responses [Epinecidin-1 also have been shown to combat esponses .R. anatipestifer, it was shown that Epinecidin-1 injection produced upregulated expression of the Mn superoxide dismutase (MnSOD) gene in brain tissue [In a duck model infected by n tissue . This gen tissue .V. vulnificus showed vaccine-like effects in addition to therapeutic effects. As the surviving zebrafish after 30 days of co-treatment were re-challenged with V. vulnificus and a survival rate of 45% was observed, while this rate was about 3% in the control group [There is evidence of AMPs adjuvant properties that have made them suitable candidates for further studies. Adjuvant-like properties of AMPs have been reported for the Epinecidin-1 in several studies . The co-ol group .Similar properties have also been reported for the advantages of using Epinecidin-1 in vaccine production. As mentioned in section 4, co-injection of Epinecidin-1 with JEV virus prevented the death in mice. Interestingly, with the re-challenge of surviving mice of the previous step, all mice survived. While the unvaccinated control group did not survive. In the study of cytokines, it was found that Epinecidin-1 induced higher levels of Th2 cytokine than Th1 cytokine . SimilarE. coioides exhibited excellent inhibition of bacterial growth against V. vulnificus [A report in 2012 showed that 30-day oral administration of Epinecidin-1 in two marine models of zebrafish and lnificus .Enterococcus faecalis, Escherichia coli, P. aeruginosa, S. aureus, Propionibacterium acnes, and C. albicans, compared to the control. It has also been reported that parameters such as low temperature and pH have no significant effect on the Epinecidin-1 bactericidal activity in cleaning solutions. Therefore, it is possible to consider this AMP for combination with various disinfectants in the future [Cleaning solutions have broad applications in community hygiene. Usually, various antimicrobial agents are added to increase the microbicidal effects, such as pyrophosphate, zinc citrate, silicon oil, benzalkonium chloride, and cocamidopropyl betaine (CAPB). Unfortunately, it has been observed that the addition of these compounds could correlate with the emergence of resistant strains to antibiotics and disinfectants. Further research, therefore, is required to test new compounds , 60. Epie future .The common property of all AMPs is to have lethal effects against one or more types of microbes . A factor that promotes the superiority of an AMP over others is a robust multi-functional activity. Regarding this issue, a strong bactericidal AMP with other activities such as immunomodulatory and anticancer features can be considered for research as an appropriate drug candidate . In addiNew and on-going findings associated with Epinecidin-1 encouraged us to write a comprehensive review of all previous studies on this peptide with high therapeutic potential to pave the way for further studies.Epinecidin-1 is a cationic AMP with an alpha-helical structure. Similar to other cationic properties (structure and function), alpha-helical AMPs can absorb to the bacterial surface due to the positive charge. The structure and positive charge made this AMP very similar to LL-37 and Pexiganan .Investigation of exact anticancer mechanisms on a broader range of cancer cells.Assess the possibility of producing topical drugs for the treatment of various infectious wounds, in particular, drug-resistant types.Helicobacter pylori effects [This peptide has very powerful anti- effects . So, it AMPs can have several functions, some of which have not been investigated for Epinecidin-1 yet. Therefore, evaluating other features such as antioxidant, anti-insect, protease inhibitor, spermicidal, and antitoxin effects can be considered.A more comprehensive review of the toxicity and side effects of this peptide, especially on the intact animal.Although many studies have been performed on this peptide, some characteristics are still not fully understood. The authors suggest the following arguments to be considered in future studies:Epinecidin-1 is a strong multi-functional AMP with seven known functions including antibacterial, antifungal, antiviral, antiprotozoal, anticancer, immunomodulatory, and wound healing activities. This peptide has high potential for further research and application in new drugs.